Genome-Wide Analysis of the Relationship between Transcriptional Regulation by Rpd3p and the Histone H3 and H4 Amino Termini in Budding Yeast

DNA in eukaryotes is packaged into chromatin by the histone proteins. The four core histones, H2A, H2B, H3, and H4, have amino-terminal “tails” that extend from the structured interior and that are subject to numerous posttranslational modifications (7, 54). Acetylation and deacetylation of the histone H3 and H4 amino termini are particularly important for gene regulation, but basic questions about the mechanism by which they affect transcription remain unanswered. For instance, a variety of enzymes can acetylate or deacetylate the H3 and/or H4 tails with different specificities, but it is not known whether modifications of specific lysine residues vary in importance at particular promoters or whether acetylation activates transcription by removing a repressive influence (unacetylated histone tails) or by providing a required positive stimulus (e.g., providing a binding surface for general transcription factors) (1). Furthermore, histone-modifying enzymes may also posttranslationally target other, nonhistone targets, complicating analysis of their functions (6, 22, 43). In an effort to understand how histone modifications affect gene regulation, we have focused in this study on histone deacetylase Rpd3p from the budding yeast Saccharomyces cerevisiae. This protein was first identified as a histone deacetylase in a screen for rat proteins binding to trapoxin, a histone deacetylase inhibitor; sequence analysis then showed it to be a homolog of the known yeast corepressor Rpd3p (46). Subsequent work demonstrated that the histone deacetylase activity of Rpd3p is needed for it to function as a corepressor and that promoter-bound histones show decreased acetylation upon recruitment of Rpd3p to nearby sites (18, 20, 35). The loss of Rpd3p results in a global increase in the acetylation of lysine residues 5, 8, 12, and 16 of histone H4 and lysine residues 9 or 18 and 14 of histone H3 (32, 34, 50), whereas promoter-bound nucleosomes near sites of Rpd3p recruitment have been reported in various studies to show the deacetylation of principally K5 of histone H4 (35); K5 and K12 of H4 and undetermined sites of histone H3 (20); and K5, K8, and K12 of H4, K9, K14, K18, K23, and K27 of H3, K27 of histone H2A and, to somewhat lesser extent, K11 and K16 of histone H2B (45). Rpd3p affects the regulation of a substantial number of genes in S. cerevisiae (3). It can be recruited to gene promoters by the repressor Ume6p but evidently by other mechanisms as well (19, 23, 32), and it is also responsible for a lower level of untargeted histone deacetylation throughout the genome in S. cerevisiae (50). Chromatin immunoprecipitation experiments have shown that repression by Rpd3p is associated with reduced levels of promoter-bound TATA binding protein, SWI/SNF, and SAGA components, and it has been suggested that reduced acetylation of histone amino termini could weaken the interactions of components of SWI/SNF and/or SAGA with promoter-bound nucleosomes, resulting in decreased levels of transcription (9, 41). However, other histone-modifying enzymes have been shown to target nonhistone proteins with functional consequences (22, 43), and it is possible that Rpd3p does so as well. A prediction that has not yet been tested is that if the histone amino termini are the principal functionally important targets of Rpd3p, then the deletion of Rpd3p should have a reduced (or no) effect in their absence. A similar approach was used previously to test the importance of the histone H3 and H4 amino termini for regulation by Gcn5p in S. cerevisiae (53). Here we report experiments testing this prediction at the gene-specific and genome-wide levels. We also compare, by microarray analysis, the roles of the H3 and H4 amino termini in gene regulation at the genome-wide level and examine the extent to which the loss of the H3 and H4 tails and the mutation of lysines to glutamines results in overlapping phenotypes with respect to transcriptional regulation. Our results provide a global view of the roles of the H3 and H4 amino termini in gene regulation in S. cerevisiae and their relationship to regulation by Rpd3p.